Extraction and Analysis of Carotenoids from Escherichia coli in Color Complementation Assays

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The Plant Journal
May 2015



A common method to investigate the function of genes putatively involved in carotenoid biosynthesis is the so called color complementation assay in Escherichia coli (see, e.g., Cunningham and Gantt, 2007). In this assay, the gene under investigation is expressed in E. coli strains genetically engineered to synthesize potential carotenoid substrates, followed by analysis of the pigment changes in the carotenogenic bacteria via high-performance liquid chromatography (HPLC). Two crucial steps in this method are (i) the quantitative extraction of the carotenoids out of E. coli and (ii) the reproducible and complete separation of the pigments by HPLC.

Here, we present a protocol for the extraction and analysis of carotenoids with a broad range of polarities from carotenogenic E. coli. The solvent mixture used for extraction keeps both the lipophilic carotenes and the more polar xanthophylls in solution and is compatible with the eluent gradient of the subsequent HPLC analysis. The C30-column used is particularly suitable for the separation of various cis-isomers of carotenoids, but also for separation of stereoisomers such as α- and β-carotene or lutein and zeaxanthin.

Keywords: Carotenoid biosynthesis (类胡萝卜素生物合成), Color complementation (颜色互补), E. coli (大肠埃希杆菌), Pigment extraction (色素提取), Carotenes (胡萝卜素), Xanthophylls (叶黄素), HPLC (HPLC), C30-column (C30柱)

Materials and Reagents

  1. 50 ml screwcap polypropylene (PP) tubes, sterile, pyrogen-free (SARSTEDT, catalog number: 62.547.254 )
  2. 15 ml screwcap PP tubes, sterile (SARSTEDT, catalog number: 62.554.502 )
  3. Escherichia coli, strain depending on the plasmids used (see Notes)
  4. Methanol HPLC grade (EMD Millipore, catalog number: 106018 )
  5. Acetone p.a. grade or higher (EMD Millipore, catalog number: 100014 )
  6. Dichloromethane p.a. grade or higher (Fisher Scientific, catalog number: 10626642 )
  7. ddH2O
  8. Ammonium acetate p.a. grade (EMD Millipore, catalog number: 101116 )
  9. Acetonitrile HPLC grade (EMD Millipore, catalog number: 113358 )
  10. Ethyl acetate HPLC grade (Merck Millipore, catalog number: 113353 )
  11. Lysogeny broth medium (Carl Roth, catalog number: X968.3 )
  12. Eluents (see Recipes)
    1. Eluent A
    2. Eluent B
    3. Eluent C


  1. For pigment extraction:
    1. Photometer (Eppendorf, model: Bio Photometer 6131 )
    2. Vortexer (Scientific Industries, model: G-560E )
    3. Ultrasonic bath (Elma Schmidbauer, model: Transsonic T 460 , or BANDELIN electronic, model: Sonorex TK52 ) (see Notes)
    4. Centrifuge (Beckman Coulter, model: Avanti J-25 )
    5. Centrifuge rotor for 50 ml tubes (Beckman Coulter, model: JA25.50 ) with round bottom adapters for 15 ml tubes (Corning, model: 8441 )
  2. For HPLC analysis:
    1. HPLC system with quaternary pump, in-line degasser, thermostatted autosampler, column heater and photodiode-array (PDA) detector (Waters, models: 2795 Separation Module and 2996 photodiode-array detector )
    2. C30-Column 250 x 4.6 mm (5.0 µm, 250 x 4.6 mm) (Bischoff, model: ProntoSIL 200-5-C30 , catalog number: 2546H300PS050)
    3. Guard column 20 x 4.0 mm (5.0 µm, 20 x 4.0 mm) (Bischoff, model: ProntoSIL 200-5-C30 , catalog number: 6302H300PS050) connected to column by guard cartridge holder (Bischoff, catalog number: 1502 0508 )


  1. HPLC-Software (Waters: Empower Pro)


  1. Bacterial culture
    1. For growth of bacteria, use sterile 50 ml PP tubes (loosely screwed cap) with 15 ml LB-medium for an optimal volume/surface ratio and oxygen supply. Inoculate with freshly transformed E. coli.
    2. Let the bacterial culture grow at 28 °C and 250 rpm to an OD600 between 0.5 and 1.0. This usually takes between 10 and 16 h, but may vary depending on the carotenogenic plasmid used. If the culture has become too dense, it may be diluted to an OD600 of 0.1 and further incubated until the recommended OD is reached. To avoid photoisomerization of carotenoids accumulating in E. coli, culture growth and pigment extraction should be performed under dim light or red light.

  2. Cell harvesting and pigment extraction
    1. Transfer 10 ml of the culture to a 15 ml PP tube, harvest the bacteria by centrifugation (4,000 x g, 4 °C, 4 min) and discard the supernatant by pouring out.
    2. Spin the bacteria-containing PP tubes again (11,000 x g, 4 °C, 1 min) and remove the remaining supernatant by pipetting. Make sure there is no residual LB medium (Figure 1A gives an example of the resulting bacterial pellet).
    3. Place the sample on ice to avoid thermal isomerization of carotenoids.
    4. Add 200 µl prechilled methanol on the pellet and resuspend the cells completely by repeated vortexing (10 sec) and sonication (10 sec) in an ultrasonic bath (see Notes) with intermittent cooling on ice. To avoid warming of the sample, the successive vortexing and sonication cycles should not take longer than 10 sec before putting the tubes back on ice.
    5. Add 200 µl prechilled acetone and mix by vortexing and sonication as described for step B4.
    6. Add 200 µl prechilled dichloromethane and mix again by vortexing and sonication as described for step B4.
    7. Incubate the tube on ice for 2 min. If the (turbid) sample appears homogenous, proceed with step B8; if there is a phase separation, add 50 µl methanol, mix by vortexing and repeat step B7 (see Figure 1 for examples of a homogenous phase [panel B] and unwanted phase separation [panel C] due to incomplete removal of LB medium in step B2).
    8. Spin the tube (11,000 x g, 4 °C, 4 min) and transfer 400 µl of the colored supernatant containing the solved pigments to an HPLC vial (Figure 1D shows a sample after centrifugation). Make sure not to disturb the pellet.

      Figure 1. Illustration of different stages of the extraction procedure. A. Bacterial pellet after step B2 (cell harvest by centrifugation); B. Turbid sample resulting in step B7 (extraction of pigments by sonication and vortexing); C. Unwanted phase separation that may occur in step B7 if LB medium was not completely removed in step B4; D. Colored supernatant and colorless pellet after step B8 (clearing extract by centrifugation).

  3. Separation and analysis of carotenoids
    1. For HPLC analysis, use the following parameters: Column temperature 28 °C; sample temperature 10 °C; injection volume 200 µl; flow 1.3 ml/min, standard detection wavelength 440 nm.
    2. Before the first measurement, the column should be equilibrated for 10 min at 28 °C with 10% eluent A and 90% eluent B (1.3 ml/min).
    3. The carotenoids are separated by applying a ternary gradient with linear changes according to Table 1.
    4. Between successive runs, equilibrate the column for 7 min with 10% eluent A and 90% eluent B (1.3 ml/min). To avoid sample carryover, washing of the injection needle with 100% methanol between HPLC runs is recommended (this is a default procedure of most autosamplers; if you inject manually using a Hamilton syringe, flush the syringe at least three times directly after each sample injection).
    5. For identification of the most common carotenoids that may be synthesized in color complementation assays using E. coli, compare the retention time and absorbance spectra of the detected pigments with the examples provided here (Figures 2 and 3 in section ‘Representative data’) or use Britton et al. (2004) as reference.

      Table 1. Ternary gradient used for HPLC analysis of extracted carotenoids. Eluent composition is given in section ‘Recipes’. The flow is 1.3 ml/min, eluent changes are linear.
      Time [min]
      Eluent A [%]
      Eluent B [%]
      Eluent C [%]

Representative data

Figure 2. Representative HPLC chromatograms of pigment extracts from carotenogenic strains of E. coli. TOP10 cells were transformed with plasmids containing different combinations of carotenoid biosynthetic genes from bacteria and algae, grown in liquid culture to an OD600 between 0.5 and 1.0, harvested and extracted as described in the protocol, and the pigment extracts analyzed on a C30 column using the HPLC gradient specified in Table 1. Average retention times of the pigments are given in parentheses. Chromatograms are normalized to the peak with the highest absorbance. Note that the relative retention of the pigments (and even their order of elution) may differ considerably between C30 and C18 columns!

Figure 3. Online PDA spectra of some of the most common carotenoids that may be synthesized in color complementation assays using E. coli. Average retention times of the pigments using a C30 column and the HPLC gradient as specified in Table 1 are given in parentheses. Absorbance spectra are normalized to the maximum absorbance.


  1. The choice of a suitable E. coli strain depends on the type of carotenogenic plasmid(s) used and the type of carotenoids that are to be accumulated. Many carotenogenic plasmids are based on pACYC184 (New England Biolabs) as backbone (Misawa et al., 1995; Cunningham and Gantt, 2007). With these plasmids, some authors observed the highest production of carotenoids in TOP10 or TOP10F’ cells (Wurtzel et al., 1997; Cunningham and Gantt, 2007), while others reported contradictory results, recommending SURE cells instead (Chae et al., 2010). We prefer TOP10, because this strain (contrary to most other popular E. coli strains) is not an amber suppressor, thus preventing improper termination of translation for genes with UAG (amber) stop codons. An in depth-discussion on the best combination of strains and plasmids for color complementation assays in E. coli is beyond the scope of this protocol. For an overview on color complementation assays using carotenoid-accumulating strains of E. coli, readers are referred to Cunningham and Gantt (2007).
  2. Carotenoids are likely to form isomers when exposed to bright light and elevated temperature. To avoid isomerization artifacts, the samples should be protected from light and heat during extraction.
  3. For sonication, a standard ultrasonic bath (volume of 3 L with a fixed sonication frequency of 35 kHz and between 120 and 240 W power) as used for cleaning procedures is recommended.
  4. In step B7, phase separation after the addition of dichloromethane only occurs if the extraction solvent is too polar. In most cases, this is caused by incomplete removal of residual LB medium in step B2. Take care to remove all remaining LB medium without disturbing the pellet.
  5. If the OD of the bacterial cultures at the projected time of harvesting is still low (OD of 0.1-0.3), the amount of solvents used in steps B4-B6 can be scaled down to 100 µl each. In step B8, transfer only 150 µl to the HPLC vial and inject only 100 µl.
  6. When stored in the autosampler at 10 °C in the dark, the pigments should remain stable and in solution for at least 6 h (we verified this for lycopene, α-carotene and β-carotene).
  7. Most HPLC methods published for C30 columns apply a binary or ternary gradient using methyl tert-butyl ether (MTBE) instead of ethyl acetate as the strongest eluent. The ternary gradient introduced here is based on eluents commonly used for C18 columns (see e.g., Kraay et al., 1992) and enables switching between C30 and C18 columns without replacement of any eluent. When using a different HPLC gradient, it is critical to verify compatibility of the extraction mixture used in this protocol with the eluent composition applied during sample injection.
  8. If pigment changes are to be analyzed quantitatively, PDA signals at the detection wavelength must not exceed the linear range of the detector. Otherwise, repeat HPLC analysis with a lower injection volume or a suitable dilution of the respective sample.


  1. Eluents
    1. Eluent A: 85% (v/v) methanol, 15% ddH2O, 0.075 M ammonium acetate
    2. Eluent B: 90% (v/v) acetonitrile, 10% ddH2O
    3. Eluent C: 100% ethyl acetate


A short version of this protocol has been published in Blatt et al. (2015). We thank the Carl-Zeiss-Stiftung for financial support.


  1. Blatt, A., Bauch. M. E., Pörschke, Y. and Lohr, M. (2015). A lycopene β-cyclase/lycopene ε-cyclase/light-harvesting complex-fusion protein from the green alga Ostreococcus lucimarinus can be modified to produce α-carotene and β-carotene at different ratios. Plant J 82(4): 582-595.
  2. Britton, G., Liaaen-Jensen, S., Pfander, H., Mercadante, A. Z. and Egeland, E. S. (2004). Carotenoids Handbook. Birkhäuser.
  3. Chae, H. S., Kim, K. H., Kim, S. C. and Lee, P. C. (2010). Strain-dependent carotenoid productions in metabolically engineered Escherichia coli. Appl Biochem Biotechnol 162(8): 2333-2344.
  4. Cunningham, F. X., Jr. and Gantt, E. (2007). A portfolio of plasmids for identification and analysis of carotenoid pathway enzymes: Adonis aestivalis as a case study. Photosynth Res 92(2): 245-259.
  5. Kraay, G. W., Zapata, M. and Veldhuis, M. J. W. (1992). Separation of chlorophylls c1, c2, and c3 of marine phytoplankton by reversed-phase-C18-high-performance liquid chromatography. J Phycol 28(5): 708-712.
  6. Misawa, N., Satomi, Y., Kondo, K., Yokoyama, A., Kajiwara, S., Saito, T., Ohtani, T. and Miki, W. (1995). Structure and functional analysis of a marine bacterial carotenoid biosynthesis gene cluster and astaxanthin biosynthetic pathway proposed at the gene level. J Bacteriol 177(22): 6575-6584.
  7. Wurtzel, E. T., Valdez, G. and Matthews, P. D. (1997). Variation in expression of carotenoid genes in transformed E. coli strains. Biores J 1: 1-11.


调查涉及类胡萝卜素生物合成的基因的功能的常见方法是在大肠杆菌中所谓的颜色互补测定(参见,例如,Cunningham和Gantt,2007) 。在该测定中,所研究的基因在E中表达。基因工程改造以合成潜在的类胡萝卜素底物,然后通过高效液相色谱(HPLC)分析色素变性细菌。该方法的两个关键步骤是(i)从E中定量提取类胡萝卜素。大肠杆菌和(ii)通过HPLC重现和完全分离颜料。

关键字:类胡萝卜素生物合成, 颜色互补, 大肠埃希杆菌, 色素提取, 胡萝卜素, 叶黄素, HPLC, C30柱


  1. 50 ml螺旋盖聚丙烯(PP)管,无菌,无热原(SARSTEDT,目录号:62.547.254)
  2. 15 ml螺旋PP管,无菌(SARSTEDT,目录号:62.554.502)
  3. 取决于所使用的质粒的大肠杆菌菌株(参见注释)
  4. 甲醇HPLC级(EMD Millipore,目录号:106018)
  5. 丙酮类年级以上(EMD Millipore,目录号:100014)
  6. 二氯甲烷年级以上(Fisher Scientific,目录号:10626642)
  7. ddH 2 O
  8. 醋酸铵等级(EMD Millipore,目录号:101116)
  9. 乙腈HPLC级(EMD Millipore,目录号:113358)
  10. 乙酸乙酯HPLC级(Merck Millipore,目录号:113353)
  11. 溶菌酵母培养基(Carl Roth,目录号:X968.3)
  12. 洗脱液(参见食谱)
    1. 洗脱液A
    2. 洗脱液B
    3. 洗脱液C


  1. 颜料提取:
    1. 光度计(Eppendorf,型号:BioPhotometer 6131)
    2. Vortexer(科学工业,型号:G-560E)
    3. 超声波浴(Elma Schmidbauer,型号:Transsonic T 460,或BANDELIN electronic,型号:Sonorex TK52)(见注释)
    4. 离心机(Beckman Coulter,型号:Avanti J-25)
    5. 用于50ml管的离心转子(Beckman Coulter,型号:JA25.50),用于15ml管的圆底适配器(Corning,型号:8441)
  2. HPLC分析:
    1. 具有四元泵,在线脱气机,恒温自动进样器,柱加热器和光电二极管阵列(PDA)检测器(Waters,型号:2795分离模块和2996光电二极管阵列检测器)的HPLC系统
    2. C30柱250×4.6mm(5.0μm,250×4.6mm)(Bischoff,型号:ProntoSIL 200-5-C30,目录号:2546H300PS050)
    3. 保护柱20×4.0mm(5.0μm,20×4.0mm)(Bischoff,型号:ProntoSIL 200-5-C30,目录号:6302H300PS050),连接到保护筒保持器的列(Bischoff,目录号:1502 0508)


  1. HPLC软件(Waters:Empower Pro)


  1. 细菌培养
    1. 对于细菌的生长,使用无菌的50ml PP管(松散拧合的帽)与15ml LB-培养基,获得最佳体积/表面比和氧气供应。接受新鲜变换的E。大肠杆菌。
    2. 让细菌培养物在28℃和250rpm下生长至0.5-1.0之间的OD 600。这通常需要10至16小时,但可能会因使用的致癌质粒而异。如果培养物变得太致密,则可将其稀释至0.100的OD 600,并进一步培养直至达到推荐的OD。避免在E中积聚的类胡萝卜素的光异构化。大肠杆菌,培养生长和色素提取应在昏暗的灯光或红光下进行
  2. 细胞收获和色素提取
    1. 将10ml培养液转移到15ml PP管中,通过离心(4,000 x g,4℃,4分钟)收获细菌,并倾倒掉弃去上清液。
    2. 再次旋转含细菌的PP管(11,000 x g,4℃,1分钟),并通过吸移除去剩余的上清液。确保没有残留的LB培养基(图1A给出了所得细菌颗粒的实例)。
    3. 将样品放在冰上以避免类胡萝卜素的热异构化。
    4. 在颗粒上加入200μl预先烧制的甲醇,并通过在超声波浴中重复涡旋(10秒)和超声波处理(10秒)完全悬浮细胞,并在冰上间歇冷却。为了避免样品升温,连续的涡旋和超声处理循环在将管放回冰之前不应超过10秒。
    5. 加入200μl预先烧制的丙酮,并按照步骤B4所述的涡旋和超声处理进行混合
    6. 加入200μl预先冷却的二氯甲烷,再按照步骤B4所述的振荡和超声处理混合
    7. 将管在冰上孵育2分钟。如果(浑浊)样品呈现均匀性,则进行步骤B8;如果存在相分离,则加入50μl甲醇,通过涡旋混合并重复步骤B7(参见图1为均相[例B]和不需要的相分离的实例[图C],这是由于步骤中不完全去除LB培养基B2)
    8. 旋转管(11,000×g,4℃,4分钟),并将400μl含有溶解的颜料的着色上清液转移到HPLC小瓶中(图1D显示离心后的样品)。确保不要打扰沉淀。

      图1.提取程序的不同阶段的图示。A.步骤B2后的细菌沉淀(通过离心收集细胞); B.产生步骤B7的浊度样品(通过超声处理和涡旋萃取颜料); C.如果在步骤B4中没有完全去除LB培养基,则可能在步骤B7中发生的不希望的相分离; D.步骤B8后的有色上清液和无色颗粒(离心清除提取物)
  3. 分离和分析类胡萝卜素
    1. 对于HPLC分析,使用以下参数:柱温28℃;样品温度10℃;注射体积200μl;流量1.3 ml/min,标准检测波长440 nm
    2. 在第一次测量之前,应在28℃下用10%洗脱剂A和90%洗脱液B(1.3ml/min)将柱平衡10分钟。
    3. 通过应用根据表1的线性变化的三元梯度来分离类胡萝卜素
    4. 在连续运行之间,用10%洗脱剂A和90%洗脱液B(1.3ml/min)使柱平衡7分钟。为了避免样品携带,建议在HPLC运行之间用100%甲醇清洗注射针头(这是大多数自动进样器的默认程序;如果使用汉密尔顿注射器手动注射,每次样品注射后直接冲洗注射器至少三次)。
    5. 用于鉴定可以在使用E的颜色互补测定中合成的最常见的类胡萝卜素。将所检测的颜料的保留时间和吸光度光谱与本文提供的实施例(图2和3在"代表性数据"部分)或使用Britton等人进行比较。 (2004)作为参考
      洗脱液A [%]
      洗脱液B [%]
      洗脱液C [%]


将TOP10细胞用来自细菌和藻类的不同组合的类胡萝卜素生物合成基因的质粒转化,在液体培养物中生长至0.5-1.0的OD 600,收获,如方案中所述提取,并使用表1中规定的HPLC梯度在C30柱上分析颜料提取物。颜料的平均保留时间在括号中给出。色谱图归一化为具有最高吸光度的峰。请注意,颜色的相对保留(甚至其洗脱顺序)可能会在C30和C18色谱柱之间有很大差异!

图3.使用E进行的颜色互补分析中可能合成的一些最常见的类胡萝卜素的在线PDA光谱。大肠杆菌 。使用C30色谱柱的色素和表1中规定的HPLC梯度的平均保留时间在括号中给出。吸光度谱被归一化为最大吸光度。


  1. 选择合适的电子邮件大肠杆菌菌株取决于所使用的类胡萝卜素质粒和待累积的类胡萝卜素的类型。许多生物质素基于pACYC184(New England Biolabs)作为骨架(Misawa等人,1995; Cunningham和Gantt,2007)。使用这些质粒,一些作者观察到TOP10或TOP10F细胞中类胡萝卜素的产量最高(Wurtzel等人,1997; Cunningham和Gantt,2007),而另一些作者报告了矛盾的结果,建议使用SURE细胞(Chae等人,2010)。我们更喜欢TOP10,因为这种菌株(与大多数其他流行的大肠杆菌菌株相反)不是琥珀抑制因子,因此防止用UAG(琥珀)终止密码子的基因的翻译不正确地终止。深入讨论了用于色彩互补分析的菌株和质粒的最佳组合。大肠杆菌超出本协议的范围。关于使用类胡萝卜素积累菌株的颜色互补测定的概述。大肠杆菌,读者可以参考Cunningham和Gantt(2007)。
  2. 当暴露于明亮的光线和升高的温度时,类胡萝卜素很可能形成异构体。为了避免异构化的假象,提取过程中样品应避免受光和热的影响
  3. 对于超声处理,推荐使用标准超声波浴(3L的固定超声波频率为35kHz,功率为120〜240W)。
  4. 在步骤B7中,如果提取溶剂太极性,则仅在加入二氯甲烷后进行相分离。在大多数情况下,这是由于在步骤B2中残留的LB培养基不完全去除引起的。小心去除所有剩余的LB培养基,而不会干扰沉淀物。
  5. 如果在预计收获时间的细菌培养物的OD值仍然很低(OD为0.1-0.3),则步骤B4-B6中使用的溶剂量可以按比例缩小到100μl。在步骤B8中,仅将150μl转移到HPLC小瓶中,并注射100μl。
  6. 当在黑暗中10℃保存在自动进样器中时,颜料应保持稳定,并在溶液中保持至少6小时(我们验证了这一点对于番茄红素,α-胡萝卜素和β-胡萝卜素)。
  7. 用于C30色谱柱的大多数HPLC方法都使用甲基叔丁基醚(MTBE)代替乙酸乙酯作为最强洗脱液的二元或三元梯度。这里引入的三元梯度是基于通常用于C18色谱柱的洗脱液(参见例如,,Kraay等人,1992),并且能够在不更换的情况下在C30和C18色谱柱之间进行切换的任何洗脱液。当使用不同的HPLC梯度时,验证本方案中使用的萃取混合物与注射样品时应用的洗脱组分的相容性至关重要。
  8. 如果定量分析颜料变化,检测波长的PDA信号不得超过检测器的线性范围。否则重复使用相应样品的注射体积或适当稀释度的HPLC分析


  1. 洗脱液
    1. 洗脱液A:85%(v/v)甲醇,15%ddH 2 O,0.075M乙酸铵,
    2. 洗脱液B:90%(v/v)乙腈,10%ddH 2 O -/-
    3. 洗脱液C:100%乙酸乙酯


该协议的简短版本已在Blatt等人发布。 (2015)。我们感谢卡尔蔡司基金会的财务支持。


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引用:Blatt, A. and Lohr, M. (2017). Extraction and Analysis of Carotenoids from Escherichia coli in Color Complementation Assays. Bio-protocol 7(6): e2179. DOI: 10.21769/BioProtoc.2179.